Each cytoskeletal element consists of a number of proteins some of which polymerize to form filamentous structures, whereas, others bind to these structures and regulate their shape, size, assembly and disassembly. The assembly and disassembly of cytoskeletal proteins is dynamic and precisely regulated. It is becoming increasingly clear that protein phosphorylation plays an important regulatory role in this dynamic process. The native proteins comprising neurofilaments and microtubule associated proteins of neurons are phosphoproteins and their phosphorylation has been shown to be modulated by hormones. Since altered phosphorylation of some of these proteins is observed in neuropathies, there is a lot of interest in characterizing the kinases involved in their in vivo phosphorylation. We have shown that neurofilament proteins and microtubule associated proteins NAP-2 and tau are good substrates for a second messenger independent kinase termed glycogen synthase kinase-3 (GSK-3) or phosphatase activating factor (Fa). This is a novel kinase which prefers to phosphorylate proteins which have been prephosphorylated at specific sites. In this proposal we will extend our studies to fully characterize phosphorylation of neurofilament proteins and microtubule associated proteins MAP-2 and tau by Fa/GSK-3. The isolated proteins will be dephosphorylated and then used as substrates for studying the effect of prephosphorylation by second messenger-dependent kinases on their subsequent phosphorylation by GSK-3. Following phosphorylation tryptic digests will be prepared and subjected to phosphopeptide analysis by HPLC. Phosphopeptides containing GSK-3 sites will be purified and sequenced. The effect of phosphorylation by GSK-3 on the abilities of these proteins to assemble into filaments or to affect filament assembly will also be investigated. The results of this investigation will further our knowledge into the possible role of GSK-3 in cytoskeletal function and will help us design experiments for determining its role in vivo.